Discovery of <i>N</i>‑(1,3,4-Thiadiazol-2-yl)amide Derivatives as Uncompetitive Inhibitors of 6‑Phosphogluconate Dehydrogenase

6-Phosphogluconate dehydrogenase (6PGD) is a crucial enzyme in the pentose phosphate pathway and a promising target for oncotherapy. However, potent 6PGD inhibitors remain limited. Herein, we report a series of N-(1,3,4-thiadiazol-2-yl)amide derivatives as 6PGD inhibitors, via in vitro enzymatic assay-based high-throughput screening and structure–activity relationship analysis. Among them, 19n exhibited significant potency against 6PGD with an IC50 value of 5.1 ± 1.0 μM. Kinetic analysis indicated that 19n was an uncompetitive inhibitor of 6PGD. Further mechanism study revealed that 19n disrupted 6PGD oligomerization in a substrate-dependent manner. Additionally, compound 19n suppressed the proliferation of A549 and Huh7 cells. Moreover, 19n treatment could result in a decrease of NADPH and Ru-5-P production as well as DNA synthesis in A549 cells. Overall, we provide a new scaffold compound 19n which expands the chemical space of 6PGD inhibitors with a novel mechanism and is worthy of further study.